Human complement

The pseudotype vesicular stomatitis virus (VSV) neutralization assay (PsVNA) used to detect neutralizing antibodies in sera utilized a non-replicating VSV-based luciferase expressing system described previously [37 (link)]. Where indicated 5 % human complement (Cedarlane) was included in the virus: antibody mixture.

Isolated rituximab samples were diluted at designated concentrations with phosphate saline buffer. Raji cells (ATCC, CCL-86) were prepared at a density of 1.0 x 10⁶ cell/mL and plated 50 μL to each well in the 96-well plate. Diluted 40-μL rituximab samples were then added to the wells followed by incubation for 10 min at room temperature. After the incubation, 40 μL of 20-fold diluted human complement (Cedarlane, Ontario, Canada) was added to the wells. Then the plates were incubated for 2 h at 37°C. The number of living cells was determined using cell count reagent SF (Nacalai Tesque, Kyoto, Japan) according to the protocol provided by the supplier. The absorbance at 460 nm was measured by VERSAmax microplate reader. (Molecular Devices, California, USA)